Methods for expression of recombinant proteins in bacterial host are widespread and offer ease of use and purification of the recombinant product. However, use of these systems for the expression of eukaryotic proteins is often limited by problems of insolubility and lack of proper post-transcription and post-translational processing (see, e.g., U.S. Pat. No. 5,721,121, incorporated herein by reference). Thus, eukaryotic expression systems are generally used for the expression of eukaryotic proteins. In particular, the pharmaceutical biotechnology industry relies heavily on the production of recombinant proteins in mammalian cells. These recombinantly produced proteins are essential to the therapeutic treatment of many diseases and conditions. In many cases, the market for these proteins exceeds a billion dollars a year. Examples of proteins produced recombinantly in mammalian cells include erythropoietin, factor VIII, factor IX, and insulin. In addition, recombinant antibodies are often used as therapeutic agents. Clinical applications of recombinantly produced proteins, in particular antibodies, often require large amounts of highly purified proteins. Proteins are generally produced in either mammalian cell culture or in transgenic animals.
Vectors for transferring the gene of interest into mammalian cells are widely available, including plasmids, retroviral vectors, and adenoviral vectors. Retroviral vectors are widely used as vehicles for delivery of genes into mammalian cells (See e.g., Vile and Russell, British Medical Bulletin, 51:12 [1995]). However, current methods for creating mammalian cell lines for expression of recombinant proteins suffer from several drawbacks. (See, e.g., Mielke et al., Biochem. 35:2239-52 [1996]). Episomal systems allow for high expression levels of the recombinant protein, but are frequently only stable for a short time period (See, e.g., Klehr and Bode, Mol. Genet. (Life Sci. Adv.) 7:47-52 [1988]). Mammalian cell lines containing integrated exogenous genes are somewhat more stable, but there is increasing evidence that stability depends on the presence of only a few copies or even a single copy of the exogenous gene. Vectors are often unstable, resulting in a decrease in the level of protein expression over time.
Based on overall product yield, expression of recombinant proteins in animals results in higher yields, relative to expression in cell culture (See e.g., Werner et al., Arzneimittelforshcung, 48:870 [1998]; Pollock et al., J. Immunol. Methods, 231:147 [1999]). However, expression in transgenic animals is limited by methods of producing transgenic mammals, variation in production and purity, and the life span of the animal.
Thus, despite continued efforts in the field, vectors for high level, continuous expression of one or more proteins in a host cell remain needed in the art.